Mammalian target of rapamycin complex 1 (mTORC1) signaling is one of the most frequently activated oncogenic pathways, making it a promising target for cancer drug discovery efforts. Although rapamycin analogues have been approved for use in select cancers and results from active site mTOR inhibitor clinical trials have yet to be reported, a new strategy for combatting cancers with hyperactive mTOR signaling is warranted, particularly since drug resistance to these agents is a growing threat. Our laboratory seeks to challenge the paradigm of directly targeting mTORC1 or mTOR kinase activity by instead affecting downstream effectors of mTORC1, namely the eIF4E?4E-BP protein-protein interaction (PPI). These proteins represent the initiator and gate-keeper, respectively, of cap-dependent mRNA translation, the process by which oncogenic proteins such as those involved in proliferation, evasion of apoptosis, metastasis and angiogenesis, the hallmarks of cancer, are produced. This PPI is regulated by mTORC1 via phosphorylation of 4E-BP, which releases eIF4E and stimulates the initiation of cap-dependent translation. Importantly, maintenance of eIF4E?4E-BP PPI homeostasis has recently been shown to be crucial for mTOR inhibitor efficacy, and many resistance mechanisms to mTOR inhibitors involve disruption of this balance through mTORC1-independent 4E-BP phosphorylation, downregulation of 4E-BP, eIF4E amplification, and Myc overexpression which selectively activates 4E-BP phosphorylation through an unknown mechanism. Thus, we hypothesize that the eIF4E?4E-BP PPI is a druggable axis in mTOR-hyperactivated cancers and targeting of this PPI will surmount drug resistance observed with mTOR inhibitors. To probe this hypothesis, we have rationally designed 4E-BP stapled peptides to act as drug-like 4E-BP mimetics to reactivate the tumor suppressor activity of 4E-BP and inhibit cancer cell proliferation. In this proposal, we describe our plans to further develop 4E-BP stapled peptides as chemical probes targeting the eIF4E?4E-BP PPI and fully decipher their mechanism-of-action through proteomic and cellular analyses. The Specific Aims of this proposal are as follows: (1) To further develop 4E-BP stapled peptides as chemical probes targeting the eIF4E?4E-BP PPI; (2) To identify and validate the cellular targets of 4E-BP stapled peptides; and (3) To determine the effect of 4E- BP stapled peptides in Myc-driven cancer cells. From these proposed studies, we will provide validated chemical probes for targeting the eIF4E?4E-BP PPI and preliminary data regarding its status as a promising therapeutic approach for the treatment of cancer.